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New Mammalian Two-Hybrid System Detects Protein-Protein Interactions

em first developed by Dr. Dang and colleagues8 provides an excellent alternative to the yeast-based system because this assay is performed in mammalian cells. Since mammalian proteins are more likely to retain their native conformation in a mammalian host, the results would more likely represent biologically significant interactions.

The Mammalian Two-Hybrid System

Fig.1

To determine the capability of the mammalian two-hybrid assay kit for studying protein-protein interactions, we constructed a set of user-friendly GAL4 BD (binding domain)-bait and NF-kB AD (activation domain)-target plasmids. These vectors were used to validate the system with known pairs of interacting proteins in various mammalian cell lines. The working principle of the mammalian two-hybrid assay kit is similar to that used in Fields yeast two-hybrid system, except that the assay detects transcriptional activation of a reporter gene (such as luciferase, SEAP, or b-galactosidase) located downstream of the multiple GAL4 DNA-binding sites. The GAL4 BD-bait, NF-kB AD-target, and a reporter plasmid are cotransfected together into a mammalian cell. The protein of interest, or bait, is expressed as a fusion protein with the GAL4 DNA binding domain, and the target protein is expressed with the activation domain. If the bait and target proteins interact, the activation domain is brought together with the DNA binding domain and together they activate transcription of the reporter gene (Figure 1).

The Mammalian Two-Hybrid Vectors

Fig.2

The pCMV-BD and pCMV-AD vectors are designed for the construction and
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